Alteration of gallium biodistribution using indium complexes for enhanced early imaging

ABSTRACT

Radiopharmaceutical compositions and methods for tumor tomography in mammals are disclosed, wherein radioactive gallium and non-radioactive indium are injected into said mammals whereby the affinity of said radioactive gallium for non-tumor tissues is decreased, resulting in enhanced imaging characteristics.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a diagnostic nuclear medicine. Morespecifically, the present invention relates to tumor imaging utilizingradioactive gallium.

2. Description of the Prior Art

Gallium-67, as the carrier-free citrate, is used routinely in clinicalmedicine in the diagnosis, staging and monitoring of several neoplasticdisease states (Semin Nucl Med 6: 331-334, 1976). Unfortunately, thisagent is plagued with imaging problems which are related to plasmaprotein binding. Specifically, gallium has an affinity for blood andsoft tissue proteins, and when given in very low doses, is extensivelybound (J Pharmacol Exp Therapeut 168: 193-198, 1973). Gallium also hasan affinity for tumors (Science 167:289-290, 1970; J Nucl Med 10:103-105, 1969) and abscesses (J Nucl Med 21: 484-488, 1980). Afterintravenous administration, gallium activity in tumors is seenimmediately and reaches a maximum within eighteen hours (Semin Nucl Med8: 193-203, 1978). Biologic clearing of gallium from blood and softtissue binding sites is a slow process, and results in the presence ofimage obscuring activity long after the ideal imaging time. Three majorconsequences of this binding phenomenon are: (a) it is necessary todelay imaging procedures for long periods of time (48-72 hours) to allowfor the clearance of non-productive radioactivity; (b) the long waitingperiod allows the build-up of image obscuring activity in the intestinaltract due to secretion of gallium into the large bowel (J Nucl Med 14:208-214, 1973); and (c) prolonged retention of large quantities of ⁶⁷ Gain the body results in a relatively high radiation dose (J Nucl Med 12:755-756, 1973).

Several reports have described methods or agents which attempt toenhance the process of imaging a tumor or abscess by shortening thebiologic half life of carrier-free gallium-67 citrate. Generally, thesefall into two categories: (a) agents which compete with radioactivegallium for blood and tissue binding sites, thus releasing theradioactive metal ion making it available for kidney elimination; or (b)complexing or chelating agents which compete with blood and tissuebinding sites for free gallium, forming a complex which is rapidlycleared by the kidney.

The initial work took the first approach and used a group (IIB) metalcomplex (Scandium citrate) as a selective competitive binding agent(South Med J 66: 1339-1340, 1973; J Nucl Med 21: 361-365, 1980).Scandium, by occupying gallium binding sites on blood proteins, preventsthe accumulation of gallium on these sites. Scandium appears to havelittle or no effect upon gallium binding in tumors. When used in animalstudies, where gallium-67 and scandium citrates were injectedsimultaneously, extremely high tumor to blood and tumor to tissue ratioswere obtained. Unfortunately, scandium produces a severe hemolyticanemia when administered to man (South Med J 66: 1339-1340, 1973; J NuclMed 21: 361-365, 1980). The use of iron dextran in similar fashion toclear gallium from rabbits bearing induced abscesses resulted in theclearing of gallium with enhanced abscess uptake of gallium (J Nucl Med17: 356-358, 1976). This ion, however, has no clinical application dueto the large dose of iron required (J Nucl Med 21: 421-424, 1980). Morerecently, it has been shown that relatively large doses ofnon-radioactive gallium will decrease blood activity withoutsignificantly reducing tumor activity (J Nucl Med 20: 656, 1979; InvestRadiol 14: 482-492, 1979).

Several investigators have used the second approach, employing achelating agent, desferoxamine, to reduce gallium blood activity (J NuclMed 21: 421-425, 1980; J Nucl Med 20: 248-251, 1979; Radiology 131:775-779, 1979; Radiology 130: 241-244, 1979). The resulting galliumdesferoxamine complex is eliminated via the kidney at a rate greaterthan that observed for gallium alone. Unfortunately, this clearing agentmust be administered 24 hours after the gallium injection in order tominimize loss of tumor activity.

SUMMARY OF THE INVENTION

It is, therefore, a primary object of the present invention to provideimproved radiopharmaceutical compositions and methods for tumor imagingwhich overcome or reduce the above mentioned problems of the prior art.

Carrier-free solutions of gallium-67 citrate were administered to normaland tumored rats, and blood levels of radioactivity were monitored as afunction of time. Metal complexes of aluminum, gallium, indium orthallium were administered at times varying from two to six hoursfollowing the injection of the radiopharmaceutical. In the cases ofindium and gallium, the blood activity was observed to decrease veryrapidly, and a generalized clearing of soft tissue activity wasobserved. Similar studies using imaging techniques, reveal that activitybound within tumors is obviously affected by the addition of moderatedoses of carrier gallium. When indium citrate was administered two hoursafter the radiopharmaceutical the tumor activity increasedsignificantly. When carrier-free gallium-67 was administeredsimultaneously with indium citrate, the biodistribution of gallium inrats bearing transplanted tumors (lymphoma and hepatoma) was found to bedramatically altered. Two hours after dosing, radioactivity is observedin the kidney, bladder, bone and tumor. Other areas and tissues wereessentially void of activity. This rapid clearance of non-productivegallium from diffuse soft tissues, blood and organs such as liver, lungand spleen demonstrates the potential for use of the gallium-68 coupledwith emission tomography.

Therefore, in accordance with the present invention, it has beendiscovered that intravenous injection of a non-radioactive indiumcomplex simultaneously with, shortly before or shortly after, theintravenous injection of carrier free gallium-67(⁶⁷ Ga) or gallium-68(⁶⁸Ga), alters the biodistribution of the radioactive gallium. Morespecifically, the indium complex occupies non-tumor gallium receptorsites, and, therefore, the gallium will not be retained on thesereceptor sites to thereby obscure the imaging process. In other words,the indium complex has a high affinity for non-tumor gallium receptorsites leaving the gallium free to be bound to the tumor gallium receptorsites.

It has been shown that indium complexes alter gallium-67 biodistributionin rats bearing transplanted tumors (lymphoma and hepatoma).

The indium and gallium complexes may be administered by injection, withintravenous injection being preferred. The gallium complexes must beadministered in carrier-free form with doses in the range of about 0.25to 500 ng being preferred. In humans, doses up to 3 mg may be employed.The indium complexes may be administered in doses of from about 0.1 to0.7 mg with doses of from about 0.1 to 0.3 mg being preferred. In humansdoses will be based on blood protein content, and may vary up to 50 mg.

All water-soluble forms of indium such as water-soluble salts may beused. These water soluble forms include water-soluble organic salts,water-soluble inorganic salts as well as various water-solublecomplexes. Examples of water-soluble organic salts include indiumcitrate, indium lactate, indium tartrate, indium phytate, and indiumphthallate. Examples of water-soluble inorganic salts are indiumchloride, indium nitrate, indium sulfate, and indium bromide. Anotheruseful form of indium is an indiumtransferrin complex.

All water-soluble forms of gallium such as water-soluble salts are alsouseful. Examples of water-soluble forms include water-soluble organicsalts, water-soluble inorganic salts as well as various water-solublecomplexes. Examples of water-soluble organic salts include galliumcitrate, gallium lactate, gallium tartrate, gallium phytate and galliumphythallate. Examples of water-soluble inorganic salts include galliumchloride, gallium nitrate, gallium sulfate and gallium bromide. Anexample of another form of gallium is a complex of gallium andtransferrin.

Radiopharmaceutical compositions may be prepared by dissolving thegallium and/or indium complex in any acceptable solvent such as water,normal saline, buffer normal to the pharmaceutical preparation, andmixtures thereof. Normal saline is preferred as a solvent.

It is preferred to administer the gallium and the indium simultaneously,however, the indium may be administered before or after the gallium,preferably within 2 hours before or after the gallium.

When gallium and indium are administered in accordance with the presentinvention the following advantages are realized: the time required forimage obscuring blood and/or soft tissue radioactivity to clear thebody, enabling one to obtain a clear image of the tumor, is reduced; theradiation dose to the patient is reduced by increasing the rate at whichthe radioactive gallium clears the body; and the short-lived positionemitting gallium-68 isotope may be recorded by emission tomographicimaging equipment to thereby allow viewing of the tumor in threedimensions. The radioactive gallium may be measured by using externalscintigraphic techniques (either photon or positron).

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the present invention will becomeapparent from a study of the following non-limiting examples anddrawings in which

FIGS. 1A-1D are distribution profiles for carrier-free gallium-67citrate:

FIG. 1A is a typical profile with no clearing agent added;

FIG. 1B is a typical profile observed when carrier gallium citrate (3.3mg/Kg) is given 6 hours (360 min) after radiopharmaceutical dosing;

FIG. 1C is a typical profile observed when indium chloride (2.1 mg/Kg)is given 6 hours (360 min) after radiopharmaceutical dosing; and

FIG. 1D is a typical profile observed when indium citrate (2.1 mg/Kg) isgiven 6 hours (360 min) after radiopharmaceutical dosing.

FIG. 2 is a rectilinear scan of a normal Sprague Dawley rat before andafter a clearing dose (3.3 mg/Kg) of non-radioactive gallium citrate wasadministered six hours post radiopharmaceutical dosing.

FIGS. 3A-3C are scans (Union Carbide Cleon model 720 large field gammacamera) of Buffalo rats bearing implanted 5123C hepatomas.

FIG. 3A shows the rats two hours after radiopharmaceutical dosing. Atthis point, the rat on the left received carrier gallium citrate (3.3mg/Kg) and the rat on the right received indium citrate (2.1 mg/Kg).

FIG. 3B shows the rats three hours after radiopharmaceutical dosing andone hour after injection of the clearing agent.

FIG. 3C shows the rats five hours past radiopharmaceutical dosing andthree hours after the clearing dose.

FIGS. 4A-4E are scans (Union Carbide Cleon model 720 large field gammacamera) of Buffalo rats, bearing Morris 5123C hepatoma is injected witha single solution containing carrier-free gallium-67 citrate and 2.1mg/Kg indium citrate.

FIG. 4A is cumulative over the time period 0 to 10 min.

FIG. 4B is the time period from 10 to 20 min.

FIG. 4C is the time period from 20 to 30 min.

FIG. 4D represents the same time period taken one hour after theinjection.

FIG. 4E represents the same time period taken two hours after theinjection.

EXAMPLES

In this study, an external blood-loop technique (Int J of Appl RadiatIsot 29: 189-190, 1978) is used to monitor blood activity of theradiopharmaceutical as a function of time. Relative tissueconcentrations are monitored by external imaging.

CHEMICALS AND RADIOPHARMACEUTICALS

All agents, both radioactive and non-radioactive, were used aspurchased, without further purification. Gallium-67 citrate was obtainedfrom Medi-Physics (Neoscan®); thallium triacetate and indium chloridewere obtained from Johnson-Mathey Chemicals Limited as the nonohydrate;and aluminum lactate was obtained from I.C.N. Pharmaceuticals.

Gallium citrate solutions were prepared by a method similar to thatdisclosed in J Am Chem Soc 72: 3822-3823, 1950. Gallium nitrate (375 mg)was dissolved in a minimum volume of boiling concentrated hydrochloricacid, citric acid solution (20% w/v, 2.5 ml) was added thereto, theresulting solution cooled to room temperature and neutralized withsodium hydroxide solution (final concentration: Ga 15 mg/ml-20 mgcitrate/ml, pH 7.4). In a similar manner, indium citrate was prepared(final concentration: In 6.67 mg/ml-20 mg citrate/ml). Aqueous solutionsof aluminum lactate (1.42 mg/ml) were obtained by solution in normalsaline. All solutions were filtered (0.22μ millipore filter) prior touse.

ANIMAL PREPARATION AND RADIOPHARMACEUTICAL DOSING

For blood clearance studies, male Sprague Dawley rats weighing 250-300grams were used. The animals were anesthetized by peritoneal injectionof an aqueous solution of urethan (1 gm/Kg). The right carotid arteryand left jugular vein were exposed and an external blood loop (PE-50tubing; volume≃0.14 ml) was inserted between the vessels. Blood wasdiverted from the artery, through the loop, and back to the animal viathe vein (Int J of Appl Radiat Isot 29: 189-190, 1978). The rightexternal jugular vein was then exposed and cannulated as an injectionsite. The animal was placed in a lead cradle and the blood loop insertedinto the shielded well of a sodium iodide.thallium crystal. The animalreceived an injection of carrier-free gallium citrate (100-120 μCi) viathe jugular cannula. Blood activity within the loop was continuouslymonitored as a function of time using a Packard Model 9012 Multi-channelAnalyzer/Multi-scaler system (Packard Instrument Co., Downersville,Ill.). Six hours post injection, the animal received a second injectioncontaining one of the clearing agents (≃7.2 mmole/Kg). In subsequentexperiments, clearing agents were administered two hours after orsimultaneously with the radioactive dose.

Initial studies used non-tumored male Sprague Dawley rats weighingbetween 250 and 300 grams. Subsequent studies in tumored rats used maleBuffalo rats bearing a 5123C hepatoma (The tumor line was initiated fromdonor animals donated by Drs. R. L. Hayes and B. Byrd of the Oak RidgeAssociated Universities). In all imaging studies, the animals wereanesthetized by intraperitoneal injection of urethane (1 gm/Kg), and theleft external jugular vein cannulated as a dosing site. The animals werethen positioned under the detection of the γ-camera (Union Carbide;CLEON 720 Large Field Gamma Camera coupled with a CLEON 110 ImageProcessor) and images were obtained periodically.

Following a single intravenous injection of carrier-free gallium-67citrate in the normal Sprague Dawley rat, prepared as described, a bloodactivity-time curve similar to that shown in FIG. 1A is observed. Thecurve is characterized by a rapid distribution phase followed by aslower elimination phase. The rapid distribution process appears to becomplete four to six hours after dosing and is in itself multiplastic.There is a very rapid component (t_(1/2) =12 seconds) which may be dueto kidney elimination of the citrate complex. This process would becomeinsignificant as gallium complexed with citrate is transferred tobinding sites on blood proteins, thus becoming less available for kidneyelimination.

The effect of a relatively large dose of non-radioactive gallium (3.3mg/Kg) given six hours after the initial injection is seen in FIG. 1B.There is a dramatic, rapid drop in blood activity (approximately 40% ofthe blood activity is removed). This is followed by a rise in bloodactivity, possibly due to release of ⁶⁷ Ga from less available galliumstores by an isotopic exchange process. The rise in blood activity peaks1.5 to 2 hours after the second injection and subsequent galliumelimination parallels that observed when no clearing agent is used. Whengallium is administered in this manner, the blood activity measuredeight hours post radiopharmaceutical dosing, is approximately 25% lessthan that observed when no clearing agent is given.

In similar experiments, equimolar quantities of non-radioactivealuminum, indium, or thallium complexes were injected six hours afterthe carrier-free gallium dose. The results of these studies aresummarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Re1ative ability of the group (IIIA) metals to clear                          .sup.67 Ga from blood six hours after injection of the radiopharmaceuti-      cal                                                                           Non-                                                                          radioactive                                                                             % Decrease in                                                                              % Incrcase in                                                                              Net                                       Metals    Blood Activity                                                                             Blood Activity                                                                             Decrease                                  ______________________________________                                        Thallium  0            0            0                                         Aluminum  9            3            6                                         Gallium   40           15           25                                        Indium (Cl)                                                                             85           25           60                                        ______________________________________                                    

There was no change in blood activity following a dose of 7.2 mmoles/Kgof thallium triacetate. Aluminum lactate caused a reduction of 9% inblood activity followed by a 3% rise in activity. The net reduction inblood activity was only 6%. Indium chloride proved to be the most potentof the clearing agents tested. As was the case with non-radioactivegallium, indium chloride (FIG. 1C) caused a rapid drop in blood activity(85%) followed by an increase which peaked 1.5 to 2 hours after theclearing dose was given. The net reduction in blood activity was 60%.When indium citrate was used in similar fashion, the blood curve wasobserved to fall 77% and there was no significant rise in blood activity(FIG. 1D). The distributional difference between indium chloride andindium citrate could be explained if citrate offered a weak, transientbinding for that gallium which is displaced from the blood proteinbinding sites now occupied by indium. The gallium citrate complex thusformed must be rapidly cleared by the kidney. This is the same mechanismsuggested above for the rapid drop in blood activity seen at earlytimes. In the absence of citrate, gallium would seek alternate,non-vascular, binding sites (probably surface proteins in tissues) fromwhich it would undergo a slower redistribution to blood sites vacated asindium undergoes distribution processes.

The two most successful clearing agents, gallium and indium citrate,were furher investigated to determine if varying the length of timebetween radiopharmaceutical dosing and clearing agent dosing altered theresults. Gallium (3.3 mg/Kg) cleared 78% of the blood activity whengiven at two hours after the radiopharmaceutical. This was followed bythe characteristic rise in activity which resulted in a net clearance of70%. Indium citrate reduced the blood activity by 78% and no subsequentrise in activity was observed.

Preliminary imaging studies performed in non-tumored rats injected withone of the four clearing agents six hours post dosing, gave resultswhich generally paralleled the blood studies. Injections of thallium oraluminum yielded no significant redistribution of the radioactivity.Injections of non-radioactive gallium or indium citrate shifted theradioactivity out of the blood/soft tissue regions and into the kidneysand the bladder (FIG. 2).

Clearing studies were performed on tumored rats using these agents indosages suggested as optimal in both blood and preliminary imagingstudies. Indium citrate was dosed at the level of 2.1 mg/Kg, and galliumcitrate was dosed at 3 mg/Kg and 15 mg/Kg. When given six hours afterthe initial injection, the high dose of non-radioactive gallium resultedin obvious redistributions of radioactivity which would have obscuredchest and abdominal images (i.e. liver, spleen, lung and intestine).However, tumor activity was observed to decrease. The lower dose ofgallium generally has a less dramatic effect in that a great deal ofactivity remains in the abdomen, particularly in those organs with highreticuloendothelial (RE) activity (liver and spleen). Also, loss ofactivity from the tumor is much less. Indium is obviously the superioragent, reducing activity within the RE rich tissues while not depletingtumor activity. However, the effect is not dramatic enough to indicateclinical significance.

When the clearing agent was given two hours post radiopharmaceuticaldosing, there were significant changes in gallium-67 distribution (FIG.3). Gallium citrate (3 mg/Kg) causes some clearing in the upper abdomenand in regions of the lower abdomen. There was little loss of activityfrom the tumor region and the diagnostic potential of the image wasimproved. The results from use of indium citrate were somewhat moredramatic in that the activity is greatly diminished throughout the softtissue regions. Activity in the liver-spleen-lung region is greatlyreduced as it is in the lower abdomen. The most significantdistributional change, however, is the obvious increase in tumoractivity. This suggests that the mechanisms by which gallium localizesin tumor must differ from that directing its localization in othertissues and blood. Further, this suggests that indium, like scandium,may allow tumor localization of activity while preventing theaccumulation of activity in blood and tissue when given simultaneouslywith the radiopharmaceutical. To test this hypothesis, indium citratewas given with gallium-67 citrate as a single solution. As shown in FIG.4, the distributional pattern of gallium-67 is drastically altered.Twenty minutes after the mixed dose is given, there is little activityoutside the kidneys, bladder, tumor and bone. After two hours, there isessentially no activity in the animal other than in the above mentionedregions.

Indium citrate, has the ability to successfully compete with gallium forblood and soft tissue binding sites, yet it does not compete for galliumbinding sites in tumor. This is a property shared with scandium andiron. It is potentially a clinically significant finding in thatgallium-67 citrate, under the distributional influence of this typeagent could be useful for general metastatic searches. No longer wouldthe liver-spleen region, a common metastatic site, be obscured byactivity taken up by the RE system. Further, because of the reduced timebetween dosing and imaging, significant abdominal secretion does notoccur, thus the gut area is free of activity.

Unlike scandium, indium is not known to rupture red blood cells.Toxicity studies following single doses of indium citrate indicate thatthe doses used in this study for clearing gallium are in the toxic range(Proc Soc Exp Biol Med 29: 1188-1193, 1931; Cancer Chemother Rep Part 159: 599-610 1975; J Indust Hyg Tox 24; 243-254, 1942). However,preliminary blood studies in normal rats suggest that clearing dose ofindium citrate can be reduced by as much as an order of magnitudewithout loss of clearing potential. Indium doses in this range were notreported to be toxic.

The use of indium citrate simultaneously with the radiopharmaceuticalcould shorten the dose-to-image waiting period from day to minutes,making possible the use of the shorter lived isotope gallium-68 (t_(1/2)=68 minutes) for tumor imaging. This isotope, while biologicallyidentical to gallium-67, has the added advantage of positron emission,introducing the potential for emission tomography and all the inherentadvantages of three dimensional imaging.

The invention being thus described, it will be obvious that the same waybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A radiopharmaceutical composition,comprising:radioactive gallium, non-radioactive indium and apharmaceutically acceptable carrier or diluent.
 2. A radiopharmaceuticalcomposition according to claim 1, wherein said radioactive gallium isgallium 67 or gallium
 68. 3. A radiopharmaceutical composition accordingto claim 1, in the form of an injectable solution.
 4. Aradiopharmaceutical composition according to claim 3, wherein saidgallium and indium are present in the form of water-soluble salts orcomplexes.
 5. A radiopharmaceutical composition according to claim 4,wherein the concentration of said indium in said injectable solution isbetween 0.3 mg/ml and 2.1 mg/ml.
 6. A radiopharmaceutical compositionaccording to claim 5, wherein the said gallium in said injectablesolution is in carrier-free form.
 7. A radiopharmaceutical diagnostickit, comprising:an effective tumor imaging amount of water-solubleradioactive gallium; and an effective tumor imaging improving amount ofwater-soluble non-radioactive indium.
 8. A method for detecting tumorsin mammals comprising:administering to said mammals an effective tumorimaging amount of radioactive gallium and an effective tumor imagingimproving amount of non-radioactive indium and measuring thebiodistribution of said radioactive gallium using external scintigraphictechniques.
 9. A method according to claim 8, wherein said gallium isadministered by injection.
 10. A method according to claim 9, whereinsaid indium is injected in an amount of between 0.1 mg/Kg and 2.1 mg/Kg.11. A method according to claim 9, wherein said gallium is injected incarrier-free quantities.
 12. A radiopharmaceutical composition accordingto claim 1, wherein said composition includes a water-soluble organicsalt of radioactive gallium.
 13. A radiopharmaceutical compositionaccording to claim 1, wherein said composition includes a water-solubleorganic salt of non-radioactive indium.
 14. A radiopharmaceuticalcomposition according to claim 1, wherein said composition includesradioactive gallium citrate.
 15. A radiopharmaceutical compositionaccording to claim 1, wherein said composition includes non-radioactiveindium citrate.
 16. A pharmaceutical composition according to claim 1,wherein said composition includes a water-soluble inorganic salt ofradioactive gallium.
 17. A pharmaceutical composition according to claim1, wherein said composition includes a water-soluble inorganic salt ofnon-radioactive indium.
 18. A radiopharmaceutical composition accordingto claim 1, wherein said composition includes an organic salt ofradioactive gallium selected from the group consisting of galliumcitrate, gallium lactate, gallium tartrate, gallium phytate and galliumphythallate.
 19. A radiopharmaceutical composition according to claim 1,wherein said composition includes an inorganic salt of radioactivegallium selected from the group consisting of gallium chloride, galliumnitrate, gallium sulfate and gallium bromide.
 20. A radiopharmaceuticalcomposition according to claim 1, wherein said composition includes acomplex of radioactive gallium and transferrin.
 21. Aradiopharmaceutical composition according to claim 1, wherein saidcomposition includes an organic salt of non-radioactive indium selectedfrom the group consisting of indium citrate, indium lactate, indiumtartrate, indium phytate and indium phthallate.
 22. Aradiopharmaceutical composition according to claim 1, wherein saidcomposition includes an inorganic salt of non-radioactive indiumselected from the group consisting of indium chloride, indium nitrate,indium sulfate and indium bromide.
 23. A radiopharmaceutical compositionaccording to claim 1, wherein said composition includes anon-radioactive indium-transferrin complex.
 24. A radiopharmaceuticalcomposition, consisting essentially of:an effective tumor imaging amountof radioactive gallium, an effective tumor imaging improving amount ofnon-radioactive indium; and a pharmaceutically acceptable carrier ordiluent.